Project Summary Endometriosis is an inflammatory disorder that affects up to 1 in 10 women of reproductive age, which is characterized by the attachment and growth of endometrial cells outside the uterus on the walls of the pelvic cavity and organs. This condition affects 50-60% of women with chronic pelvic pain and may be found in up to 50% of infertile women. Along with dysmenorrhea (painful periods), endometriosis creates incapacitating physical symptoms, such as severe nonmenstrual pain, painful sex, and gastrointestinal pain. Asymptomatic endometriosis is a leading cause of infertility. Despite its prevalence and myriad negative impacts on quality of life, endometriosis often goes undiagnosed. One major reason for delayed diagnosis is that no reliable non- invasive test currently exists, and laparoscopic surgery is the standard procedure for confirmatory diagnosis. DotLab has identified circulating microRNA biomarkers that are both sensitive and specific for endometriosis, forming the basis for a non-invasive test with enormous potential for transforming clinical practice. MicroRNAs (miRNAs) are short, noncoding RNA molecules that post-transcriptionally regulate target genes, and their altered expression has been associated with a multitude of diseases. Through a comprehensive microarray screen of serum from women with and without endometriosis, DotLab has discovered ten miRNAs that are significantly up- or down-regulated in endometriosis and can be readily quantified in serum and saliva. In both retrospective and prospective clinical studies, combinations of three or four of these miRNAs yielded a high diagnostic value, with sensitivity and specificity >90% to distinguish endometriosis from other benign gynecological diseases. Two of the main hurdles for translating the test into clinical practice are: 1) demonstrating the generalizability of the biomarkers to identify endometriosis across diverse locations, patient populations, and stages of the disease; and 2) determining the ability of these biomarkers to indicate whether mild or severe endometriosis is present. In Phase I we have outlined a research strategy to surmount these challenges: by 1) validating the performance of our miRNA biomarkers in a multi-center prospective study, in women from different geographic areas and ethnic backgrounds; and 2) conducting a pilot study to determine which of our miRNA biomarkers show the strongest correlation with two independent, quantitative metrics of endometriosis severity (clinical: pain symptom scores, and pathological: bulk of disease). Successful completion of this Phase I SBIR project will confirm the utility of our non-invasive biomarker test and will pave the way for Phase II studies to support our in-vitro diagnostic (IVD) regulatory submission. Results from Phase I will also be critical to help determine the sample size needed for Phase II longitudinal studies to monitor disease severity in women undergoing different endometriosis therapies. Clinical use of DotLab's non-invasive biomarkers could dramatically improve patient care by enabling medical providers to diagnose patients sooner, begin therapy earlier, and guide treatment decisions for the millions who suffer from the condition.